U.S. patent number 9,617,529 [Application Number 14/416,539] was granted by the patent office on 2017-04-11 for method for production of recombinant human alpha-galactosidase a.
This patent grant is currently assigned to JCR PHARMACEUTICALS CO., LTD.. The grantee listed for this patent is JCR Pharmaceuticals Co., Ltd.. Invention is credited to Masahiro Asano, Tsuyoshi Fukui, Yukichi Hatano, Atsuko Kawasaki, Kazutoshi Mihara, Atsushi Sugimura, Toshihiro Yagi.
United States Patent |
9,617,529 |
Asano , et al. |
April 11, 2017 |
Method for production of recombinant human alpha-galactosidase
A
Abstract
Disclosed is a method for production of recombinant human
alpha-galactosidase A (rh alpha-Gal A) in a large scale, with a
high purity. The method comprises the steps of (a) culturing rh
alpha-Gal A-producing mammalian cells in a serum-free medium, (b)
collecting culture supernatant, (c) subjecting the culture
supernatant to anion-exchange column chromatography, (d) to
hydrophobic column chromatography, (e) to a column chromatography
employing as solid phase a material having affinity for phosphate
group, (f) to cation-exchange column chromatography, and (g) to gel
filtration column chromatography, in the order.
Inventors: |
Asano; Masahiro (Hyogo,
JP), Yagi; Toshihiro (Hyogo, JP), Fukui;
Tsuyoshi (Hyogo, JP), Kawasaki; Atsuko (Hyogo,
JP), Hatano; Yukichi (Hyogo, JP), Mihara;
Kazutoshi (Hyogo, JP), Sugimura; Atsushi (Hyogo,
JP) |
Applicant: |
Name |
City |
State |
Country |
Type |
JCR Pharmaceuticals Co., Ltd. |
Hyogo |
N/A |
JP |
|
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Assignee: |
JCR PHARMACEUTICALS CO., LTD.
(Hyogo, JP)
|
Family
ID: |
49996710 |
Appl.
No.: |
14/416,539 |
Filed: |
July 24, 2013 |
PCT
Filed: |
July 24, 2013 |
PCT No.: |
PCT/JP2013/004510 |
371(c)(1),(2),(4) Date: |
January 22, 2015 |
PCT
Pub. No.: |
WO2014/017088 |
PCT
Pub. Date: |
January 30, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20150210992 A1 |
Jul 30, 2015 |
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Foreign Application Priority Data
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Jul 26, 2012 [JP] |
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PCT/JP2012/004776 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N
9/2465 (20130101) |
Current International
Class: |
C12N
9/24 (20060101); C12N 9/40 (20060101); A61K
38/16 (20060101) |
Field of
Search: |
;435/208 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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94/12628 |
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Jun 1994 |
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WO |
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98/11206 |
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Mar 1998 |
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WO |
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00/11353 |
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Mar 2000 |
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WO |
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00/53730 |
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Sep 2000 |
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WO |
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2011/108451 |
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Sep 2011 |
|
WO |
|
Other References
Brady et al., "Replacement Therapy for inherited enzyme
deficiency," N. Engl. J. Med., vol. 289, No. 1, pp. 9-14 (1973).
cited by applicant .
Desnick et al., "Enzyme therapy in Fabry disease: differential in
vivo plasma clearance and metabolic effectiveness of plasma and
splenic alpha-galactosidase A isozymes," Proc Natl Acad Sci USA,
vol. 76, No. 10, pp. 5326-5330 (1979). cited by applicant .
Mapes et al., "Enzyme replacement in Fabry's disease, an inborn
error of metabolism," Science, vol. 169, pp. 987-989 (1970). cited
by applicant .
Bishop et al., "Human alpha-galactosidase: characterization and
eukaryotic expression of the full-length cDNA and structural
organization of the gene," Lipid Storage Disorders, pp. 809-823
(1988). cited by applicant .
Bishop et al., "Human alpha-galactosidase A: nucleotide sequence of
a cDNA clone encoding the mature enzyme," Proc Natl Acad Sci USA,
vol. 83, pp. 4859-4863 (1986). cited by applicant .
International Search Report for PCT/JP2013/004510, dated Sep. 3,
2013, 6 pages total. cited by applicant .
Written Opinion of the International Searching Authority for
PCT/JP2013/004510, dated Sep. 3, 2013, 4 pages total. cited by
applicant.
|
Primary Examiner: Monshipouri; Maryam
Attorney, Agent or Firm: Hamre, Schumann, Mueller &
Larson, P.C.
Claims
The invention claimed is:
1. A method for production of a recombinant human alpha-Gal A
comprising the steps of: (a) culturing recombinant human alpha-Gal
A-producing mammalian cells in a serum-free medium to let them
secrete the recombinant human alpha-Gal A in the medium, (b)
collecting culture supernatant by removing the cells from the
culture that is obtained in step (a) above, (c) subjecting the
culture supernatant collected in step (b) above to an
anion-exchange column chromatography to collect first recombinant
human alpha-Gal A-active fractions, (d) subjecting the first
fractions collected in step (c) above to a hydrophobic column
chromatography to collect second recombinant human alpha-Gal
A-active fractions, wherein the hydrophobic column chromatography
employs a hydrophobic ligand, wherein the hydrophobic ligand
contains a phenyl group, (e) subjecting the second fractions
collected in step (d) above to a column chromatography employing as
solid phase a material having affinity for phosphate group to
collect third recombinant human alpha-Gal A-active fractions, (f)
subjecting the third fractions collected in step (e) above to a
cation-exchange column chromatography to collect fourth recombinant
human alpha-Gal A-active fractions, and (g) subjecting the fourth
fractions collected in step (f) above to a gel filtration column
chromatography to collect fifth recombinant human alpha-Gal
A-active fractions, in the order listed.
2. The method according to claim 1, wherein the cation exchanger
employed in the cation-exchange column chromatography is a weak
cation exchanger.
3. The method according to claim 2, wherein the weak cation
exchanger has a selectivity based on both hydrophobic interaction
and hydrogen bond formation.
4. The method according to claim 2, wherein the weak cation
exchanger has phenyl groups, amide bonds and carboxyl groups.
5. The method according to claim 1, wherein the material having
affinity for phosphate group is selected from the group consisting
of hydroxyapatite and fluoroapatite.
6. The method according to claim 5, wherein the material having
affinity for phosphate group is hydroxyapatite.
7. The method according to claim 1, wherein the mammalian cells are
CHO cells transfected with an expression vector which is designed
to express the recombinant human alpha-Gal A under the regulation
of EF-1(alpha) promoter.
8. The method according to claim 1, wherein, in the step (d), the
column employed in the hydrophobic column chromatography is
equilibrated with a phosphate buffer supplemented with a salt, and
wherein, in step (d), the recombinant human alpha-Gal A is eluted
with a phosphate buffer not supplemented with a salt.
9. The method according to claim 8, wherein the salt is sodium
chloride.
10. The method according to claim 9, wherein a concentration of the
sodium chloride is 500 mM to 1 M.
Description
TECHNICAL FIELD
The present invention relates to a method for production of
recombinant human alpha-galactosidase A (rh alpha-Gal A). More
specifically the present invention relates to a process for
production of rh alpha-Gal A by culturing rh alpha-Gal A-producing
mammalian cells in a serum-free medium, as well as to a process for
purification of rh alpha-Gal A obtained in the culture supernatant
through column chromatography, in high yield and to such a high
purity as permitting direct use of the purified protein as a
medical drug.
BACKGROUND ART
Alpha-galactosidase A (alpha-Gal A) is a lysosomal enzyme having an
activity to hydrolyze the terminal alpha-galactosyl bond of
glycolipids and glycoproteins. Trihexosylceramide, which contains
three hexose moieties, is one of the substrates for
alpha-galactosidase A and receives hydrolysis at its terminal
hexose moiety by the enzyme. Human alpha-Gal A is expressed as a
precursor peptide comprising 429 amino acids, of which N-terminal
31 amino acids constitute a signal peptide. The precursor is
processed to the mature peptide, which consists of 398 amino acids
left behind after the removal of the signal peptide, and a pair of
the mature peptide molecules readily form a homodimer, wherever
condition allows, which is the enzymatically active form of human
alpha-Gal A. Human alpha-Gal A possesses oligosaccharide chains
containing mannose-6-phosphate residues and can be targeted to the
lysosome via a mannose-6-phosphate receptor.
Fabry's disease is an X-linked inherited lysosomal storage disease,
which is caused by inherited abnormality of gene encoding alpha-Gal
A. Lack of the activity of this enzyme causes accumulation of
trihexosylceramide in various tissues, resulting in renal
impairment, angiokeratomas, and cardiovascular abnormalities,
including ventricular enlargement and mitral valve
insufficiency.
It had already been known in the 1960's that the enzymatic activity
of alpha-galactosidase A is scarcely or only weakly detected in the
tissues of patients with Fabry's disease. It thus had been
speculated that some inherited abnormality of the gene encoding
alpha-Gal A should be the cause of Fabry's disease. Based on this
speculation, alpha-Gal A replacement therapy for Fabry's disease
had been conducted by infusing alpha-Gal A extracted and purified
from human plasma or spleen to the patient, resulting in the
confirmation that the alpha-Gal A replacement therapy could lower
the levels of trihexosylceramide in the blood of the patients
suffering from Fabry's disease (see NPL 1, NPL 2, NPL 3). Therefore
it had been considered that the alpha-Gal A enzyme replacement
therapy should be a promising therapy for Fabry's disease.
Practical application of the alpha-Gal A replacement therapy,
however, has been extremely limited due to the lack of supply of
this enzyme.
In 1986, the gene encoding human alpha-Gal A was isolated (see NPL
4), and further, in 1988, a method was reported for expression and
production of rh alpha-Gal A by using CHO cells transformed with a
human alpha-Gal A expression vector (see NPL 5). Utilizing
recombinant technology, these finding made it possible to produce
human alpha-Gal A in a large scale, and to use the enzyme as a
medicament for the enzyme replacement therapy for Fabry's disease.
The rh alpha-Gal A has also been expressed in insect cells (PTL
1).
Further, some methods for purification of rh alpha-Gal A were
reported comprising employment of hydrophobic column
chromatography, heparin sepharose column chromatography,
hydroxylapatite column chromatography, anion-exchange column
chromatography, and gel filtration column chromatography (PTL 2,
PTL 3). Although the purity of the rh alpha-Gal A obtained by the
method for purification described in PTL 2 or PTL 3 has shown to be
sufficiently high, it was not considered to be fully acceptable for
human use. The reason is that while any medicaments to be infused
to patients are required to be free of contaminants, such as, in
particular, proteins derived from the host cells employed, either
PTL 2 or PTL 3 fails to show whether such contaminants remain in
the purified rh alpha-Gal A or not. Currently, medicaments against
Fabry's disease which contain rh alpha-Gal A as an active
ingredient have been on the market as medical drugs under the trade
names such as Replagal.TM. and Fabrazyme.TM..
CITATION LIST
Patent Literature
[PTL 1] WO 90/11353
[PTL 2] WO98/11206
[PTL 3] WO00/53730
Non Patent Literature
[NPL 1] Mapes et al: Science 169: 987-9 (1970)
[NPL 2] Brady et al: N. Engl. J. Med. 289: 9-14 (1973)
[NPL 3] Desnick et al: Proc Natl Acad Sci USA 76: 5326-30
(1979).
[NPL 4] Bishop et al: Proc Natl Acad Sci USA 83: 4859-63 (1986)
[NPL 5] Bishop et al: Lipid Storage Disorders--Biological and
Medical Aspects, NY Plenum Press P809-823 (1988)
SUMMARY OF INVENTION
Technical Problem
Against the above background, it is an objective of the present
invention to provide a method for production of recombinant human
alpha-galactosidase A (rh alpha-Gal A) starting with culturing of
rh alpha-Gal A producing mammalian cells in a serum-free
medium.
It is another object of the present invention to provide a process
for purification of rh alpha-Gal A obtained in the culture
supernatant, through column chromatography, in high yield and to
such a high purity as permits direct use of the purified protein as
a medical drug.
Solution to Problem
The present inventors found that rh alpha-Gal A contained in the
supernatant of a culture of rh alpha-Gal A-producing cells in a
serum-free medium, can be purified to a very high purity, and in a
very high yield as well, by subjecting the rh alpha-Gal A to a
purification process comprising a combination of anion-exchange
column chromatography, hydrophobic column chromatography,
chromatography using a column having affinity for phosphate group,
cation-exchange column chromatography, and gel filtration column
chromatography. Alternatively, anion-exchange column chromatography
above may be substituted by dye affinity column chromatography. The
present invention was completed through further studies based on
the finding.
Thus, the present invention provides what follows:
1. A method for production of a recombinant human alpha-Gal A
comprising the steps of: (a) culturing recombinant human alpha-Gal
A-producing mammalian cells in a serum-free medium to let them
secrete the recombinant human alpha-Gal A in the medium, (b)
collecting culture supernatant by removing the cells from the
culture that is obtained in step (a) above, (c) subjecting the
culture supernatant collected in step (b) above to anion-exchange
column chromatography or dye affinity column chromatography to
collect the recombinant human alpha-Gal A-active fractions, (d)
subjecting the fractions collected in step (c) above to hydrophobic
column chromatography to collect recombinant human alpha-Gal
A-active fractions, (e) subjecting the fractions collected in step
(d) above to a column chromatography employing as solid phase a
material having affinity for phosphate group to collect the
recombinant human alpha-Gal A-active fractions, (f) subjecting the
fractions collected in step (e) above to cation-exchange column
chromatography to collect the recombinant human alpha-Gal A-active
fractions, and (g) subjecting the fractions collected in step (f)
above to gel filtration column chromatography to collect the
recombinant human alpha-Gal A-active fractions, in the order.
2. The method according to (1) above, wherein the cation exchanger
employed in the cation-exchange column chromatography is a weak
cation exchanger.
3. The method according to (2) above, wherein the weak cation
exchanger has a selectivity based on both hydrophobic interaction
and hydrogen bond formation.
4. The method according to (2) or (3) above, wherein the weak
cation exchanger has phenyl groups, amide bonds and carboxyl
groups.
5. The method according to one of (1) to (4) above, wherein the dye
employed in the dye affinity column chromatography is a blue
triazine dye.
6. The method according to one of (1) to (5) above, wherein the
material having affinity for phosphate group is selected from the
group consisting of hydroxyapatite and fluoroapatite.
7. The method according to (6) above, wherein the material having
affinity for phosphate group is hydroxyapatite.
8. The method according to one of (1) to (7) above, wherein the
mammalian cells are CHO cells transfected with an expression vector
which is designed to express the recombinant human alpha-Gal A
under the regulation of EF-1(alpha) promoter.
9. A method for purification of a recombinant human alpha-Gal A
having a mannose 6-phosphate residue in its oligosaccharide chain,
and wherein the method comprises the steps of: (a) applying the
recombinant human alpha-Gal A with contaminants to a chromatography
column which employs as solid phase a material having affinity for
phosphate group, (b) flowing a first mobile phase through the
column to wash the column while letting the recombinant human
alpha-Gal A be adsorbed by the column, and (c) eluting the
recombinant human alpha-Gal A from the column by flowing a second
mobile phase through the column, wherein the second mobile phase
contains a phosphate at higher concentration than the first mobile
phase.
10. The method according to (9) above, wherein the material having
affinity for phosphate group is selected from the group consisting
of hydroxyapatite and fluoroapatite.
11. The method according to (10) above, wherein the material having
affinity for phosphate group is hydroxyapatite.
12. A recombinant human alpha-Gal A composition produced by the
method according to one of (1) to (11).
Advantageous Effects of Invention
As it has enabled to produce rh alpha-Gal A starting with
serum-free culturing of cells, the present invention provides rh
alpha-Gal A which is free of any serum-derived contaminants,
including pathogenic agents such as viruses or prions. Therefore,
the rh alpha-Gal A obtained according to the present invention can
be administered into a human body as a safe medicament
substantially without any risks of exposure to such pathogenic
agents. Further, as it has enabled purification of rh alpha-Gal A
substantially avoiding any use of organic solvents, the present
invention eliminates the risk of denaturation of rh alpha-Gal A
which otherwise might be caused by the exposure to an organic
solvent employed. Furthermore, the present invention is favorable
to the environment, for the waste fluid left after performing the
purification process according to it does not contain an organic
solvent, and in the economic sense as well, for it requires no
facility to treat organic solvents which would otherwise be
contained in the waste fluid.
Further the present invention makes it possible to selectively
purify rh alpha-Gal A having mannose 6-phosphate residue in its
oligosaccharide chain. To exert its enzymatic activity after
administration to a human body, the rh alpha-Gal A should be taken
up by relevant cells through mannose 6-phosphate receptors
expressed on their surface. Therefore the efficacy of the rh
alpha-Gal A as a medicament is dramatically increased through the
selective purification of it according to the present
invention.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows a schematic diagram illustrating the method for
construction of vector pE-neo/hGHpA.
FIG. 2-1 shows a first part of a schematic diagram illustrating the
method for construction of vector pE-gs/hGHpA(alpha-Gal A).
FIG. 2-2 shows a second part of a schematic diagram illustrating
the method for construction of vector pE-gs/hGHpA(alpha-Gal A).
FIG. 3 shows a growth curve of recombinant cells for expression of
rh alpha-Gal A and the concentration of rh alpha-Gal A in the
culture medium.
FIG. 4 shows the result of SDS-PAGE electrophoresis of the
fractions obtained in the respective purification steps. In each
lane, 5 micrograms of protein was applied. Lane 1: Molecular size
marker, Lane 2: concentrated culture supernatant, Lane 3:
Q-sepharose Fast Flow fraction, Lane 4 Phenyl sepharose Fast Flow
fraction, Lane 5: Hydroxyapatite column (CHT-I) faction, Lane 6:
CaptoMMC fraction, Lane 6: Superdex 200 prep grade column
fraction.
FIG. 5 shows the SE-HPLC chart of the purified rh alpha-Gal A (Lot
No. 1). The vertical and horizontal axes show absorbance at 215 nm
and retention time, respectively.
DESCRIPTION OF EMBODIMENTS
In the present invention, while the recombinant human
alpha-galactosidase A (rh alpha-Gal A) is preferably a recombinant
protein of human wild-type alpha-Gal A, it is not excluded that the
rh alpha-Gal A is a mutant-type protein having one or more
substitution, deletion, and/or insertion of one or more amino acids
compared with the amino acid sequence of human wild-type alpha-Gal
A. The DNA sequence for human wild-type alpha-Gal A and amino acid
sequence encoded by it, including N-terminal signal sequence, are
shown as SEQ ID NO:13 and SEQ ID N0:14, respectively. The
N-terminal signal sequence consists of 31 amino acids and is
removed post-translationally.
In the present invention, the term "recombinant human alpha-Gal
A-producing mammalian cells" or "rh alpha-Gal A-producing mammalian
cells" means mammalian cells which have been artificially
manipulated to express, or strongly express, the gene encoding
human alpha-Gal A. Though in general the gene to be strongly
expressed is one which is introduced to the mammalian cells
(transformation) using an expression vector in which the gene is
incorporated, it may also be an intrinsic gene which has been
artificially modified to be strongly expressed. Examples of the
means for artificially modifying an intrinsic gene to make it
strongly express itself include, but not limited to, replacement of
the promoter upstream of the intrinsic gene with a promoter which
strongly induces expression of the gene. Such methods have been
disclosed in several literatures (i.e. WO94/12650, WO95/31560).
Though there is no particular limitation as to which mammalian
cells are to be employed, preferred are those of human-, mouse- or
Chinese hamster-origin, and, among others, CHO cells, which
originate from Chinese hamster ovary cells, are particularly
preferred.
In the present invention, the term "recombinant human
alpha-galactosidase A" or "rh alpha-Gal A" means the human
alpha-galactosidase A which is secreted by the above-mentioned
recombinant human alpha-Gal A-producing mammalian cells in the
medium during culture.
In the present invention, the term "oligosaccharide chain" means a
chain of oligosaccharides covalently bound to the peptide chain of
alpha-Gal A, including an asparagine type sugar chain, which is
covalently bound to an asparagine residue of alpha-Gal A.
In the present invention, an example of preferable serum-free media
in which the rh alpha-Gal A-producing mammalian cells are to be
cultured is the following medium which comprises; 3-700 mg/mL of
amino acids, 0.001-50 mg/L of vitamins, 0.3-10 g/L of
monosaccharides, 0.1-10000 mg/L of inorganic salts, 0.001-0.1 mg/L
of trace elements, 0.1-50 mg/L of nucleosides, 0.001-10 mg/L of
fatty acids, 0.01-1 mg/L of biotin, 0.1-20 micrograms/L of
hydrocortisone, 0.1-20 mg/L of insulin, 0.0-10 mg/L of vitamin B12,
0.01-1 mg/L of putrescine, 10-500 mg/L of sodium pyruvate, and
water-soluble iron compounds. As desired, it may also include
thymidine, hypoxanthine, a conventional pH indicator, and
antibiotics.
Further, DMEM/F12 medium, a mixed medium consisting of DMEM and
F12, may also be used as a basic serum-free medium. Both of these
media are well known to those skilled in the art. As a serum-free
medium, DMEM(HG)HAM modified (R5) medium may be used, too, which
contains sodium hydrogen carbonate, L-glutamine, D-glucose,
insulin, sodium selenite, diaminobutane, hydrocortisone, ferric
(II) sulfate, asparagine, aspartic acid, serine, and polyvinyl
alcohol. Furthermore, a commercially available serum-free medium
may also be used as a basic medium, such as CDoptiCHO, CHO-S-SFM II
or CD CHO (Invitrogen), IS CHO-V or IS CHO-V-GS (Irvine),
EX-CELL302 or EX-CELL305 (JRH), or the like.
In the present invention, rh alpha-Gal A-producing mammalian cells
are cultured for rh alpha-Gal A production in the culture medium
for at least 5 days, more preferably for 8 to 14 days. Then the
culture supernatant containing rh alpha-Gal A is collected and
provided to the purification process for rh alpha-Gal A.
Each of the chromatography procedures for purification of the rh
alpha-Gal A may, when needed, be carried out in the presence of a
nonionic surfactant in order to prevent nonspecific binding of the
protein. Though there is no particular limitation as to which
nonionic surfactant is to be employed, a polysorbate-based
surfactant is preferably employed, and more preferably polysorbate
80 or polysorbate 20. The concentration of such a nonionic
surfactant is preferably 0.005% (w/v) to 0.05% (w/v), more
preferably about 0.01% (w/v).
The process for purification of the rh alpha-Gal A may be carried
out at room temperature or at lower temperatures, but preferably at
lower temperatures, particularly at 1-10 deg C.
In the first chromatography step for the purification process, the
pH of the culture supernatant is adjusted preferably to 7.0-8.0,
more preferably to pH 7.2-7.8, and further preferably to about pH
7.5. Then the culture supernatant is applied to the anion-exchange
column that has been equilibrated with a phosphate buffer
supplemented with a salt to let rh alpha-Gal A bind to the column.
The pH of this phosphate buffer is preferably to 7.0-8.0, more
preferably about 7.2-7.8, and still more preferably about 7.5.
Though there is no particular limitation as to which salt is to be
added to the phosphate buffer, sodium chloride and potassium
chloride are preferred. In the case where sodium chloride is used,
its concentration is preferably in the range of 5-100 mM, more
preferably in the range of 10-60 mM, and still more preferably
about 50 mM.
After the anion-exchanger column to which rh alpha-Gal A is bound
is washed, the rh alpha-Gal A is eluted using a phosphate buffer
with increased salt concentration. Though there is no particular
limitation as to which salt is to be used, sodium chloride and
potassium chloride are preferred. In the case where sodium chloride
is used, its concentration is preferably in the range of 125-500
mM, more preferably 175-350 mM, and still more preferably about 225
mM.
Further, though there is no particular limitation as to which
anion-exchanger resin is to be employed, strong anion-exchanger
resin may be used preferably. Commercially available resins, such
as Q Sepharose Fast Flow (GE Healthcare), may be used
preferably.
Alternatively, the dye affinity chromatography may be used as the
first chromatography step for the purification process. In this
case the pH of the culture supernatant is adjusted preferably to
6.5-7.5, more preferably to 6.8-7.2, and still more preferably to
about 7.0, then the culture supernatant is applied to the dye
affinity column that has been equilibrated with a phosphate buffer
supplemented with a salt. The pH of this phosphate buffer is
preferably 6.5-7.5, more preferably 6.8-7.2, and still more
preferably about 7.0. Concurrently, Tris buffer may be added to the
phosphate buffer at the concentration of preferably 30-70 mM, more
preferably about 50 mM.
After the column to which rh alpha-Gal A is bound is washed, the rh
alpha-Gal A is eluted. Elution may be done by a phosphate buffer
with an increased pH. The concentration of a salt in the buffer may
be increased at the same time. The pH of this phosphate buffer is
preferably 7.0-7.8, more preferably 7.3-7.7, and still more
preferably about 7.5. In the case where the concentration of a salt
is also increased, sodium chloride and potassium chloride are
preferably used for this purpose. When sodium chloride is chosen,
its concentration is preferably in the range of 400-800 mM, more
preferably in the range of 500-700 mM, and still more preferably
about 600 mM. Concurrently, Tris buffer may be added to the
phosphate buffer at the concentration of preferably 30-70 mM, more
preferably about 50 mM.
Further, though there is no particular limitation as to which resin
is to be employed for dye affinity chromatography, blue triazine
dye is preferably used, but other triazine dyes are also suitable.
A particularly preferred column material for this is Blue Sepharose
6 Fast Flow (GE Healthcare) in which the dye, Cibacron.TM. Blue
F3GA, is covalently immobilized to Sepharose 6 Fast Flow
matrix.
The hydrophobic column chromatography, the second step of the
purification process, is a step for removing contaminants making
use of the hydrophobic interaction between rh alpha-Gal A and
hydrophobic ligand attached to the resin. Though there is no
particular limitation as to which hydrophobic ligand is to be
employed in the hydrophobic column chromatography, preferable is a
ligand containing phenyl group, and more preferable a ligand
containing phenyl group attached to the resin via a spacer arm,
such as represented by the formula
R--O--CH.sub.2--CH(OH)--CH.sub.2--O--C.sub.6H.sub.5 where R denotes
the resin. A particularly preferred column material for this is
Phenyl Sepharose 6 Fast Flow (GE Healthcare) in which phenyl group
is covalently immobilized to Sepharose 6 Fast Flow matrix via a
certain spacer arm.
The hydrophobic column chromatography column is equilibrated with a
phosphate buffer supplemented with a salt. The pH of this phosphate
buffer is preferably 6.5-7.5, more preferably about 6.7-7.3, and
still more preferably about 7.0. Though there is no particular
limitation as to which salt is to be added to the phosphate buffer,
sodium chloride and potassium chloride are preferred. In the case
where sodium chloride is used, its concentration is preferably in
the range of 500 mM-1 M, and more preferably in the range of
700-800 mM, and still more preferably about 750 mM. Concurrently,
Tris buffer may be added to the phosphate buffer at the
concentration of preferably 30-70 mM, more preferably about 50
mM.
Then, rh alpha-Gal A-containing fractions of the eluate obtained in
the first step are applied to the column. After the column to which
rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted.
Elution may be done by a phosphate buffer not supplemented with
salt, where the concentration of phosphate buffer is preferably
3-10 mM, and still more preferably about 5 mM. Concurrently, Tris
buffer may be added to the phosphate buffer at the concentration of
preferably 30-70 mM, more preferably about 50 mM. Alternatively
elution may be done by pure water.
The column chromatography employing a solid phase having affinity
for phosphate group, the third step of the purification, is a step
to eliminate contaminant proteins, such as carboxyl peptidases.
Though there is no particular limitation as to which solid phase is
to be used having affinity for phosphate group, hydroxyapatite and
fluoroapatite may preferably be used, and hydroxyapatite is
particularly preferred.
The hydroxyapatite column is equilibrated with a phosphate buffer
supplemented with a salt. The pH of this phosphate buffer is
preferably 6.5-7.5, more preferably about 6.8-7.2, and still more
preferably about 7.0, and the concentration of phosphate preferably
3.0-20 mM, more preferably about 3.0-15 mM, and further preferably
about 10 mM. Though there is no particular limitation as to which
salt is to be added to the phosphate buffer, sodium chloride and
potassium chloride are preferred. In the case where sodium chloride
is used, its concentration is preferably lower than 800 mM, and
more preferably in the range of 10-200 mM, and further preferably
about 50 mM. Concurrently, Tris buffer may be added to the
phosphate buffer at the concentration of preferably 30-70 mM, more
preferably about 50 mM.
Then rh alpha-Gal A-containing fractions of the eluate obtained in
the second step are applied to the column. After the column to
which rh alpha-Gal A is bound is washed, the rh alpha-Gal A is
eluted. Elution may be done by a phosphate buffer containing a
salt. The pH of this phosphate buffer is preferably 6.5-7.5, more
preferably about 6.7-7.3, and still more preferably about 7.0, and
the concentration of phosphate is preferably to 30-80 mM, more
preferably about more than 40-60 mM, and still more preferably
about 45 mM. Though there is no particular limitation as to which
salt is to be used, sodium chloride and potassium chloride are
preferred. In the case where sodium chloride is used, its
concentration is preferably lower than 300 mM, more preferably in
the range of 10-200 mM, and still more preferably of about 50
mM.
The cation-exchange column chromatography, the forth step of the
purification process, is a step to eliminate contaminant proteins.
Though there is no particular limitation as to which
cation-exchanger is to be employed in the cation-exchange column
chromatography, a weak cation exchanger is preferred, and more
preferred is a weak cation exchanger having a selectivity based on
both hydrophobic interaction and hydrogen bond formation. For
example, a weak cation exchanger having phenyl groups, amide bonds
and carboxyl groups and having a selectivity based on both
hydrophobic interaction and hydrogen bond formation, such as Capto
MMC (GE Healthcare), etc., may be employed
In the cation-exchange column chromatography, the column is
equilibrated with a phosphate buffer supplemented with a salt. The
pH of this phosphate buffer is preferably 4.5-6.5, more preferably
about 5.0-6.0, and still more preferably about 5.5. Though there is
no particular limitation as to which salt is to be added to the
phosphate buffer, sodium chloride, potassium chloride and calcium
chloride are preferred. In the case where sodium chloride is used,
its concentration is preferably in the range of 50-250 mM, and more
preferably in the range of 100-200 mM, and still more preferably
about 150 mM.
Then rh alpha-Gal A-containing fractions of the eluate obtained in
the third step are applied to the column. After the column to which
rh alpha-Gal A is bound is washed, the rh alpha-Gal A is eluted
using a phosphate buffer with an increased pH, which is preferably
in the range of about 5.5-7.5, more preferably in the range of
about 6.0-7.0 and still more preferably about 6.5
The gel filtration column chromatography, the fifth step of the
purification process, is a step for elimination of low
molecular-weight impurities, such as endotoxins, as well as
multimeric complexes or decomposition products of rh alpha-Gal A.
Thus, substantially pure rh alpha-Gal A is obtained through the
steps 1st to 5th.
Additionally one more column chromatography may be incorporated
into the purification process mentioned above, comprising
anion-exchange or dye affinity column chromatography, hydrophobic
column chromatography, column chromatography employing a solid
phase having affinity for phosphate group, cation-exchange column
chromatography, and gel filtration column chromatography in the
order. Such the additional column chromatography is preferably dye
affinity column chromatography and may be employed between any two
adjacent steps of the purification process mentioned above.
A step for virus inactivation may optionally be added to the
purification process of the present invention. Though there is no
particular limitation as to which process for virus inactivation is
to be applied, a solvent-detergent method may preferably be
applied. For this, a nonionic surfactant is added to a solution
containing rh alpha-Gal A, and the mixture is incubated for more
than 3 hours. Though there is no particular limitation as to which
surfactant is to be used, polysorbate 20, polysorbate 80,
tritonX-100, and tri(n-butyl)phosphate may preferably be used alone
or in any combinations, and more preferably the combination of
polysorbate 80 and tri(n-butyl)phosphate may be used.
Such an additional step for virus inactivation may be employed
either previous to the purification process or between any two
adjacent steps of the purification process mentioned above.
Further, the present invention also provides a method for
purification of rh alpha-Gal A having a mannose 6-phosphate residue
in its oligosaccharide chain. The method includes a step in which a
solution containing rh alpha-Gal A is subjected to column
chromatography employing a solid phase having affinity for
phosphate group. Preferred examples of such a solid phase include
hydroxyapatite and fluoroapatite, and hydroxyapatite is
particularly preferred. The purpose of this step employing a solid
phase having affinity for phosphate group is to purify rh alpha-Gal
A containing mannose 6-phosphate residues in its oligosaccharide
chain. The step can be performed in combination with at least one
other purification step, selected from cation-exchange column
chromatography, hydrophobic column chromatography, dye-affinity
column chromatography, anion-exchange column chromatography, and
gel filtration column chromatography, in any order. Preferably, the
combination is anion-exchange column chromatography, hydrophobic
column chromatography, column chromatography employing a solid
phase having affinity for phosphate group, cation-exchange column
chromatography, and gel filtration column chromatography in the
order.
The present invention is described in further detail below with
reference to examples. However, it is not intended that the present
invention be limited to the examples.
Example 1
Construction of pE-Gs/hGHpA(Alpha-GalA), a Rh Alpha-Gal A
Expression Vector
pEF/myc/nuc vector (Invitrogen) was digested with KpnI and NcoI to
cut out a DNA fragment including the EF-1(alpha) promoter and its
first intron, which then was blunt-ended with T4 DNA polymerase.
Then the DNA fragment thus prepared was inserted into pC1-neo
vector (Invitrogen) which had been digested with BglII and EcoRI
and then blunt-ended with T4 DNA polymerase. Inserted into this was
the above-mentioned blunt-ended fragment including the EF-1(alpha)
promoter and its first intron to give pE-neo vector (FIG. 1).
A DNA fragment containing poly adenylation signal sequence of human
growth hormone gene was synthesized by annealing four synthetic
oligonucleotides: (a) hGH-f1 (ID NO:1), (b) hGH-r1 (SEQ ID NO:2),
(c) hGH-f2 (SEQ ID NO:3), and (d) hGH-r2 (SEQ ID NO:4). The DNA
fragment thus prepared was inserted between the MluI and KpnI sites
of pE-neo to give pE-neo/hGHpA vector (FIG. 1).
A 5' half of Chinese hamster Glutamine synthetase ("Ch GS" or "GS")
coding region was amplified by PCR using a primer set, GS-1 (SEQ ID
NO:5) and GS-3 (SEQ ID NO:6), and, as a template, ssDNA prepared
from RNA extracted from CHO cells. A 3' half of Ch GS coding region
was amplified by PCR using a primer set, GS-2 (SEQ ID NO:7) and
GS-4 (SEQ ID NO:8), and, as a template, ssDNA prepared from RNA
extracted from CHO cells. Then the amplified 5' half of Ch GS
coding region was ligated into pT7blue-T vector (EMD Millipore).
The vector thus obtained was digested with EcoRV and BamHI, and
then the amplified 3' half of CH GS coding region, after digestion
with EcoRV and BamHI, was inserted into the digested vector to
obtain a plasmid vector containing the full length cDNA for Ch-GS.
The plasmid obtained was designated pT7blue(GS) (FIG. 2-1).
A 5' half of human alpha-GalA coding region was amplified by PCR
using a primer set, GAL-Mlu (SEQ ID NO:9) and GAL-Sca2 (SEQ ID
NO:10), and, as a template, human placental Quick-Clone cDNA
(Clontech). A 3' half of human alpha-GalA coding region was
amplified by PCR using a primer set, GAL-Sca1 (SEQ ID NO:11) and
GAL-Xba (SEQ ID NO:12), and, as a template, human placental
Quick-Clone cDNA (Clontech). Then both PCR products were ligated
into the EcoRV site of pBluescriptSK(+) vector. The 5' half of
human alpha-GalA coding region then was excised from the vector
thus obtained, by digestion with MluI and Sca1, and the 3' half of
human alpha-GalA coding region was excised from the vector by
digestion with Sca1 and NotI Then the excised fragments were
concomitantly inserted into the MluI and NotI sites of the multi
cloning site in pE-neo vector to obtain full length cDNA for human
alpha-GalA. The plasmid thus obtained was designated
pE-neo(alpha-GalA) (FIG. 2-2).
pE-neo/hGHpA was digested with AfIII and BstXI, and into the vector
thus digested was inserted Ch-GS cDNA which had been excised out
from pT7blue(GS) by digestion with AfIII and BstXI. The plasmid
obtained was designated pE-gs/hGHpA (FIG. 2-1). pE-gs/hGHpA vector
was digested with MluI and NotI, and into the vector thus digested
was inserted human alpha-GalA cDNA which had been excised out from
pE-neo(alpha-GalA) by digestion with MluI and NotI. The plasmid
obtained, designated pE-gs/hGHpA(alpha-GalA), was used as rh
alpha-Gal A expression vector in further experiments (FIG.
2-2).
Example 2
Production of Recombinant Cells for Expression of Rh Alpha-Gal
A
CHO cells (CHO-K1: purchased from American Type Culture Collection)
was transfected with the above-mentioned expression vector
pE-gs/hGHpA(alpha-Gal A) using Lipofectamine 2000 (Invitrogen)
according to the following method. Briefly, two days before
transfection, 2.5.times.10.sup.5 CHO-K1 cells were seeded in a
3.5-cm culture dish containing 3 mL of D-MEM/F12 medium containing
5% FCS (D-MEM/F12/5% FCS), and the cells were cultured at 37 deg C.
in a humidified atmosphere of 5% CO.sub.2 and 95% air. After two
days of culture, the cells were washed with PBS, and 1 mL of fresh
serum-free D-MEM/F12 was added. Then the cells were transfected by
addition of 200 microliters of a 1:1 mixture solution consisting of
Lipofectamine 2000 solution diluted 20 times with Opti-MEM I medium
(Invitrogen) and a pE-gs/hGHpA(alpha-GalA) solution diluted to 20
micrograms/mL with Opti-MEM I medium, followed by incubation at 37
deg C. in a humidified atmosphere of 5% CO.sub.2 and 95% air for 5
hours. After transfection, the medium was replaced with
D-MEM/F12/5% FCS and cultivation followed for 24 hours.
Then, the medium was replaced with glutamine-free GMEM-S medium
(JRH Biosciences) supplemented with 1.times.GS supplement (JRH
Biosciences), 10% dialyzed FBS and 30 micromole/L methionine
sulfoximine (MSX), and a selective culture was carried out at 37
deg C. in a humidified atmosphere of 5% CO.sub.2 and 95% air. Cells
that had grown in the medium for selective culture were subjected
to several successive rounds of subculture in the medium to give
recombinant cells. In the course of the selective culture,
concentration of MSX in the medium was elevated step by step to
amplify the inserted recombinant genes. Finally bulk cells that
stably grew in the medium with 300 micromole/L MSX were obtained
after two steps subculture at elevated MSX concentrations (100 and
300 micromole/L MSX).
Then, according to the limiting dilution technique, the cells
having the amplified recombinant gene were seeded on a 96-well
plate in such a manner that not more than one cell might be seeded
per well. The cells then were cultured for about 10 days to let
each of them form a monoclonal colony. The culture supernatant in
the wells where a monoclonal colony was formed was each sampled and
examined for human alpha-Gal A activity as described below, and
cell lines which were found expressing a high activity for human
alpha-Gal A were selected.
For adaptation to serum-free suspension cell culture, the selected
cell lines were each cultured in a commercially available
serum-free medium, CD-Opti CHO medium (Invitrogen) supplemented
with 10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine and
100 micromole/L MSX, at 37 deg C. in a humidified atmosphere of 5%
CO.sub.2 and 95% air until the cells stably grew. The cells then
were suspended in CD-Opti CHO medium (Invitrogen) supplemented with
10 mg/L insulin, 40 mg/L thymidine, 100 mg/L hypoxantine, 100
micromole/L MSX, and 10% DMSO, and stored as seed cells in liquid
nitrogen.
Example 3
Culture of Recombinant Cells for Expression of Rh Alpha-Gal A
The above seed cells were thawed and diluted to a density of
4.times.10.sup.5 cells/mL and cultured for 3 to 4 days with CD-Opti
CHO medium supplemented with 10 mg/L insulin, 40 mg/L thymidine,
100 mg/L hypoxantine and 100 micromole/L MSX (CD medium), and then
diluted to a density of 2.times.10.sup.5 cells/mL with CD medium
and cultured for 3 to 4 days. These cells were again diluted to a
density of 2.times.10.sup.5 cells/mL with CD medium and further
cultured for 3 to 4 days.
The number of the cells thus cultured was counted, and the cell
culture was diluted with CD medium to a density of 2.times.10.sup.5
cells/mL, and then the cells in 1 L of the diluted culture were
cultured with shaking for a few days. The scale of culture was
escalated until the culture volume reached 200 L.
Then the number of the cells was counted, and the cells were
diluted to a density of 2.times.10.sup.5 cells/mL with 1800 L of
CD-Opti CHO medium supplemented with 10 mg/L insulin, 40 mg/L
thymidine, 100 mg/L hypoxantine and 0.1% HY soy (Invitrogen), and
cultured for 11 days. 22.4 L of feed solution which contained 8.84
g insulin, 35.2 mmol hypoxantine, 5.6 mmol thymidine and 1.8 kg HY
soy in CD-Opti CHO medium was added on the 3rd, 5th, 7th, and 9th
days. Sampling was made every day during the culture, and
measurement was performed for cell number, survival rate, glucose
concentration, and amount of expressed rh alpha-Gal A. In the case
where glucose concentration became lower than 3 g/L, 30% glucose
solution was added so that the concentration reached 3.5 g/L.
During the 11 days of culture, the viable cell density and the
concentration of rh alpha-Gal A were daily monitored. The viable
cell density reached about 6.times.10.sup.6 cells/mL on days 6-7 of
culture, indicating that high-density cell culture was successfully
achieved (FIG. 3). As measured by ELISA as described below, the
concentration in the medium of rh alpha-Gal A secreted by the cells
increased over time and reached almost 140 mg/L on day 6 (FIG.
3).
After completion of the culture above, the cell culture was
collected and filtered successively through Millistak+ HC Pod
Filter grade DOHC, (Millipore), Millistak+ HC Pod Filter grade XOHC
(Millipore), and then Durapore Opticap XLT20 (0.22 micrometer,
Millipore), to give a culture supernatant. The obtained culture
supernatant was concentrated by a hollow fiber module equipped with
PS membrane to one 13th of the original volume.
Example 4
Virus Inactivation
Tri-n-butyl phosphate (TNBP) and polysorbate 80 were added to the
approximately 75 L of the concentrated culture supernatant
described above so that their final concentrations would be 0.3%
(v/v) and 1% (w/v), respectively, and this mixture solution then
was gently stirred for 3 to 4 hours at room temperature.
Example 5
Method for Purification of Rh Alpha-Gal A
The pH of the above virus-inactivated solution then was adjusted to
7.5 by addition of 1 M Tris-buffer (pH 8.8), and subsequently the
conductivity was adjusted to that of the 10 mM phosphate buffer (pH
7.5) containing 50 mM NaCl, by addition of 50 mM phosphate buffer
(pH 7.0) containing 1 M NaCl. Then the solution was filtered
through Durapore Opticap XL10 (Millipore), and then was loaded on a
Q Sepharose Fast Flow column (column volume: about 19.2 L, bed
height: about 20 cm, GE Healthcare), an anion-exchange column,
which had been equilibrated with 10 mM phosphate buffer (pH 7.5)
containing 50 mM NaCl at a linear flow rate of 150 cm/hr to let rh
alpha-Gal A be adsorbed by the column. Then, the column was washed
with a threefold column volume of the same buffer supplied at the
same flow rate, and the adsorbed rh alpha-Gal A then was eluted
with a sixfold column volume of 10 mM phosphate buffer (pH 7.5)
containing 225 mM NaCl, subsequently with a threefold column volume
of 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and the
fractions containing rh alpha-Gal A were collected.
To the fractions collected at the step of Q Sepharose Fast Flow
column chromatography above, one fourth volume of 2 M NaCl was
added to adjust the concentration of NaCl to 500 mM, and
subsequently acetic acid was added to adjust the pH to 7.0. The
solution obtained was loaded on a Phenyl Sepharose 6 Fast Flow
column (column volume: 19.2 L, bed height: about 20 cm, GE
Healthcare), a hydrophobic column, which had been equilibrated with
50 mM Tris-HCl, 5 mM phosphate buffer (pH 7.0) containing 750 mM
NaCl, at a linear flow rate of 150 cm/hr to let rh alpha-Gal A be
adsorbed by the column. Then after the column was washed with a
threefold column volume of the same buffer supplied at the same
flow rate, the adsorbed rh alpha-Gal A was eluted with a ninefold
column volume of a buffer containing 50 mM Tris-HCl and 5 mM
phosphate (pH 7.0), and subsequently with a threefold column volume
of pure water, and the fractions containing rh alpha-Gal A were
collected.
The fractions collected at the step of Phenyl Sepharose 6 Fast Flow
column chromatography above, was loaded on a CHT-I ceramic
hydroxyapatite column (column volume: 19.2 L, bed height: about 20
cm, Bio-Rad Laboratories), a hydroxyapatite column, which had been
equilibrated with 50 mM Tris-HCl, 10 mM phosphate buffer (pH 7.0)
containing 50 mM NaCl, at a linear flow rate of 150 cm/hr to let rh
alpha-Gal A be adsorbed by the column. Then, the column was washed
with a fivefold column volume of the same buffer supplied at the
same flow rate, and the adsorbed rh alpha-Gal A then was eluted
with a ninefold column volume of a buffer containing 50 mM
Tris-HCl, 45 mM sodium phosphate and 50 mM NaCl (pH 7.0), and
subsequently with a threefold column volume of 200 mM phosphate
buffer (pH 7.0), and the fractions containing rh alpha-Gal A were
collected.
To the fractions collected at the step of CHT-I ceramic
hydroxyapatite column chromatography above, acetic acid was added
to adjust the pH to 5.5. A precipitate formed by this was removed
by filtration through Opticap SHC XL5 (0.22 micrometer, Millipore).
The solution obtained was loaded on a Capto MMC column (column
volume: 19.2 L, bed height: about 20 cm, GE Healthcare), a
cation-exchange column having a selectivity based both on
hydrophobic interaction and on hydrogen bond formation, which had
been equilibrated with 50 mM acetate buffer (pH 5.5) containing 150
mM NaCl, at a linear flow rate of 150 cm/hr to let rh alpha-Gal A
be adsorbed by the column. Then after the column was washed with a
fourfold column volume of the same buffer supplied at the same flow
rate, the adsorbed rh alpha-Gal A was eluted with a fivefold column
volume of 50 mM phosphate buffer (pH 6.5), and subsequently with a
threefold column volume of 50 mM phosphate buffer (pH 7.0)
containing 1 M NaCl, and the fractions containing rh alpha-Gal A
were collected.
The fractions collected at the step of Capto MMC column
chromatography above was concentrated using Pellicon 3 Cassettes
equipped with Biomax.TM. 30 membrane (Millipore) until the total
volume became less than 1 L. One third of the concentrated solution
was loaded on a Superdex 200 prep grade column (column volume:
about 58 L, bed height: about 30 cm.times.2, GE Healthcare) which
had been equilibrated with 20 mM phosphate buffer (pH 5.8)
containing 137 mM NaCl. The same buffer then was supplied at a
linear flow rate of 25 cm/hr, and the fractions which corresponded
to the main peak observed by absorbance at 280 nm were collected as
fractions containing purified rh alpha-Gal A. This chromatography
was carried out three times to purify all the concentrated solution
above, and the fractions collected by each run were pooled.
The fractions pooled at the step of Superdex 200 prep grade column
chromatography above were concentrated by Hollow fiber module
equipped with PS membrane, and to the concentrated solution,
polysorbate 80 was added at the final concentration of 0.02%. The
obtained solution was filtered through Viresolve Pro Modus1.3 (0.07
m.sup.2 size, Millipore) to avoid any possible contamination with
viruses and microorganisms in the final product.
The amount of rh alpha-Gal A after each step was quantified using
ELISA as described below. The recovery rate of rh alpha-Gal A of
each purification step is shown in Table 1, in which "rh alpha-Gal
A recovery rate/step" means the proportion of the amount of
recovered rh alpha-Gal A to that of the loaded rh alpha-Gal A in
each step, and "rh alpha-Gal A recovery rate/total" the proportion
of the amount of recovered rh alpha-Gal A in each process to the
initial amount of rh alpha-Gal A subjected to the purification
process. The amount of rh alpha-Gal A subjected to the above
purification process was 133.8 g, of which 78.5 g of rh alpha-Gal A
was finally recovered, thus giving the rh alpha-Gal A recovery
rate/total as high as 58.7%. These results show that the method for
purification described above enables to purify rh alpha-Gal A in
very high yield and in a large production scale.
[Table 1]
TABLE-US-00001 TABLE 1 Recovery rate of rh alpha-Gal A in each
purification step (Lot No. 1) rh rh rh rh alpha-Gal alpha-Gal
alpha-Gal alpha-Gal A A A A recovery recovery loaded recovered
rate/step rate/total Purification process (g) (g) (%) (%)
Anion-exchange column 133.8 139.4 104.2 104.2 (Q Sepharose FF)
Hydrophobic column 123.3 112.8 91.5 84.3 (Phenyl Sepharose FF)
Hydroxyapatite column 112.2 106.6 94.1 78.9 (CHT-I) Cation-exchange
column 105.0 100.1 95.4 74.8 (Capto MMC) Gel filtration column 94.2
78.5 83.3 58.7 (Superdex 200 pg)
Example 6
Analysis of Human Alpha-Gal A by ELISA
To each well of a 96-well microtiter plate (Nunc) was added 100
microliters of mouse anti-human alpha-Gal A monoclonal antibody
diluted to 0.25 micrograms/mL with 0.05 M carbonate-bicarbonate
buffer (pH 9.6), and the plate was let stand for at least 1 hour at
room temperature or overnight at 2-8 deg C. to let the antibody be
absorbed by the wells. Then, after each well was washed with
phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20
(PBS-T), 300 microliters of PBS-T containing 1% BSA was added, and
the plate was let stand for at least one hour at room temperature.
Then, after each well was washed three times with PBS-T, 100
microliters of the test sample or human alpha-Gal A standard, which
had been diluted as desired with PBS containing 0.5% BSA and 0.05%
Tween 20 (PBS-BT), was added to the well, and the plate was let
stand for at least one hour at room temperature. Then, after each
well was washed three times with PBS-T, 100 microliters of
horseradish peroxidase-labeled rabbit anti-human alpha-Gal A
polyclonal antibody diluted with PBS-BT was added and the plate was
let stand for at least one hour. Then, after each well was washed
three times with PBS-T, 100 microliters of 0.4 mg/mL
o-phenylenediamine with phosphate-citrate buffer (pH 5.0)
containing 0.009% hydrogen peroxide was added to the well, and the
plate was let stand for 10 to 25 minutes at room temperature. Then
0.1 mL of 1 mol/L H.sub.2SO.sub.4 was added to each well to stop
the reaction, and the optical density at 490 nm was measured for
the well on a 96-well plate reader.
Example 7
Measurement of the Activity of Rh Alpha-Gal A
Standard solutions were prepared by dissolving
4-methylumbelliferone in Dilution Buffer (26.7 mM Citrate-44.8 mM
phosphate buffer, pH 4.6 containing 0.1 mg/mL BSA,) at
concentrations of 0 to 200 micromole/L. A substrate solution was
prepared by dissolving
4-methylumbelliferyl-alpha-D-galactopyranoside (SIGMA) in Dilution
Buffer at the final concentration of 5 mM. 10 microliters of each
of the standard solutions or the test sample diluted with Dilution
Buffer was added to each well of a 96-well microtiter plate
(FluoroNunc Plate, Nunc). 75 microliters of the substrate solution
was added to each well containing the diluted test sample or any of
the standard solutions, and the plate was let stand for one hour at
37 deg C. in the dark. After this incubation, 200 microliters of
Stop Buffer (0.2 M glycine-NaOH, pH 10.6) was added to each well.
Then fluorescent intensity of the solution in each well was
measured by a 96-well plate reader with excitation light at the
wavelength of 335 nm and fluorescent light at the wavelength of 460
nm.
A standard curve was produced by plotting fluorescence intensity
measured of the standard solutions and interpolating between the
plotted data points. The fluorescence intensity of each sample was
corresponded to the concentration of the released 4-MUF using the
standard curve. The specific activity of human alpha-Gal A was
calculated as Units/mg, where one Unit of activity was equal to one
micromole of 4-MUF produced per minute at 37 deg C. A published US
patent application (publication No. 2004-0229250) was referred to
for conducting the above measurement of the activity of rh
alpha-Gal A. The specific activity of rh alpha-Gal A purified by
the process described above was found to be about 50 U/mg (46.7 to
54.5 U/mg).
Example 8
Analysis of Purity of Rh Alpha-Gal A
The rh alpha-Gal A purified above was subjected to SDS-PAGE
electrophoresis under a reductive, heating (70 deg C. 10 min.)
condition. The gel-stained with Coomassie brilliant blue revealed a
single band at the position of molecular weight of about 55 kD in
Superdex 200 pg fraction (FIG. 4, lane 7).
Further, rh alpha-Gal A purified above was analyzed by
Size-elimination HPLC (SE-HPLC). HPLC was performed using LC-20A
System, SPD-20AV UV/VIS Detector (Shimazu Corp.). For SE-HPLC
analysis, 10 microliters of a sample solution containing 2 mg/mL of
rh alpha-Gal A purified above was applied onto TSKgel
G3000SW.sub.XL column (7.8 mm I.D..times.30 cm, TOSOH) equilibrated
with 25 mM phosphate buffered saline (PBS) at a flow rate of 0.5
ml/min. Elution profile was produced by monitoring absorbance at
215 nm.
The SE-HPLC analysis of the above purified rh alpha-Gal A revealed
a single peak alone (FIG. 6). The result showed that the rh
alpha-Gal A obtained above was highly purified and free of any
detectable contaminants. This result demonstrates that the rh
alpha-Gal A purified above is of such a high purity as may be
directly used as a medical drug.
INDUSTRIAL APPLICABILITY
The present invention is utilized for production of rh
alpha-galactosidase A (rh alpha-Gal A) in a large scale, with a
sufficiently high purity to be administered as a medicament to a
patient suffering from Fabry's syndrome.
SEQUENCE LISTING FREE TEXT
SEQ ID NO:1=hGH-f1, synthetic sequence
SEQ ID NO:2=hGH-r1, synthetic sequence
SEQ ID NO:3=hGH-f2, synthetic sequence
SEQ ID NO:4=hGH-r2, synthetic sequence
SEQ ID NO:5=Primer GS-1, synthetic sequence
SEQ ID NO:6=Primer GS-3, synthetic sequence
SEQ ID NO:7=Primer GS-2, synthetic sequence
SEQ ID NO:8=Primer GS-4, synthetic sequence
SEQ ID NO:9=Primer GAL-Mlu, synthetic sequence
SEQ ID NO:10=Primer GAL-Sca2, synthetic sequence
SEQ ID NO:11=Primer GAL-Sca1, synthetic sequence
SEQ ID NO:12=Primer GAL-Xba, synthetic sequence
SEQUENCE LISTINGS
1
141114DNAArtificial SequencehGH-f1, synthetic sequence 1cgcgtgaatt
cgcggccgct ctagacccgg gtggcatccc tgtgacccct ccccagtgcc 60tctcctggcc
ctggaagttg ccactccagt gcccaccagc cttgtcctaa taaa
1142124DNAArtificial SequencehGH-r1, synthetic sequence 2tgatgcaact
taattttatt aggacaaggc tggtgggcac tggagtggca acttccaggg 60ccaggagagg
cactggggag gggtcacagg gatgccaccc gggtctagag cggccgcgaa 120ttca
124396DNAArtificial SequencehGH-f2, synthetic sequence 3attaagttgc
atcattttgt ctgactaggt gtccttctat aatagcgcag caccatggcc 60tgaaataacc
tctgaaagag gaacttggtt aggtac 96478DNAArtificial SequencehGH-r2,
synthetic sequence 4ctaaccaagt tcctctttca gaggttattt caggccatgg
tgctgcgcta ttatagaagg 60acacctagtc agacaaaa 78525DNAArtificial
SequencePrimer GS-1, synthetic sequence 5cttaagatgg ccacctcagc
aagtt 25627DNAArtificial SequencePrimer GS-3, synthetic sequence
6acgatatccc tgccataggc tttgtct 27727DNAArtificial SequencePrimer
GS-2, synthetic sequence 7agacaaagcc tatggcaggg atatcgt
27830DNAArtificial SequencePrimer GS-4, synthetic sequence
8ccatcgtgat ggttagtttt tgtattggaa 30926DNAArtificial SequencePrimer
GAL-Mlu, synthetic sequence 9ggtacgcgtg acaatgcagc tgagga
261021DNAArtificial SequencePrimer GAL-Sca2, synthetic sequence
10gtgattgcag tactgtcgga t 211121DNAArtificial SequencePrimer
GAL-Sca1, synthetic sequence 11atccgacagt actgcaatca c
211235DNAArtificial SequencePrimer GAL-Xba, synthetic sequence
12gagtctagat tttaaagtaa gtcttttaat gacat 35131290DNAHomo
sapiensCDS(1)..(1290) 13atg cag ctg agg aac cca gaa cta cat ctg ggc
tgc gcg ctt gcg ctt 48Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly
Cys Ala Leu Ala Leu 1 5 10 15 cgc ttc ctg gcc ctc gtt tcc tgg gac
atc cct ggg gct aga gca ctg 96Arg Phe Leu Ala Leu Val Ser Trp Asp
Ile Pro Gly Ala Arg Ala Leu 20 25 30 gac aat gga ttg gca agg acg
cct acc atg ggc tgg ctg cac tgg gag 144Asp Asn Gly Leu Ala Arg Thr
Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 cgc ttc atg tgc aac
ctt gac tgc cag gaa gag cca gat tcc tgc atc 192Arg Phe Met Cys Asn
Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 agt gag aag
ctc ttc atg gag atg gca gag ctc atg gtc tca gaa ggc 240Ser Glu Lys
Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 tgg
aag gat gca ggt tat gag tac ctc tgc att gat gac tgt tgg atg 288Trp
Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90
95 gct ccc caa aga gat tca gaa ggc aga ctt cag gca gac cct cag cgc
336Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg
100 105 110 ttt cct cat ggg att cgc cag cta gct aat tat gtt cac agc
aaa gga 384Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser
Lys Gly 115 120 125 ctg aag cta ggg att tat gca gat gtt gga aat aaa
acc tgc gca ggc 432Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys
Thr Cys Ala Gly 130 135 140 ttc cct ggg agt ttt gga tac tac gac att
gat gcc cag acc ttt gct 480Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile
Asp Ala Gln Thr Phe Ala 145 150 155 160 gac tgg gga gta gat ctg cta
aaa ttt gat ggt tgt tac tgt gac agt 528Asp Trp Gly Val Asp Leu Leu
Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 ttg gaa aat ttg gca
gat ggt tat aag cac atg tcc ttg gcc ctg aat 576Leu Glu Asn Leu Ala
Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 agg act ggc
aga agc att gtg tac tcc tgt gag tgg cct ctt tat atg 624Arg Thr Gly
Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 tgg
ccc ttt caa aag ccc aat tat aca gaa atc cga cag tac tgc aat 672Trp
Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215
220 cac tgg cga aat ttt gct gac att gat gat tcc tgg aaa agt ata aag
720His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys
225 230 235 240 agt atc ttg gac tgg aca tct ttt aac cag gag aga att
gtt gat gtt 768Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile
Val Asp Val 245 250 255 gct gga cca ggg ggt tgg aat gac cca gat atg
tta gtg att ggc aac 816Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met
Leu Val Ile Gly Asn 260 265 270 ttt ggc ctc agc tgg aat cag caa gta
act cag atg gcc ctc tgg gct 864Phe Gly Leu Ser Trp Asn Gln Gln Val
Thr Gln Met Ala Leu Trp Ala 275 280 285 atc atg gct gct cct tta ttc
atg tct aat gac ctc cga cac atc agc 912Ile Met Ala Ala Pro Leu Phe
Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 cct caa gcc aaa gct
ctc ctt cag gat aag gac gta att gcc atc aat 960Pro Gln Ala Lys Ala
Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 cag gac
ccc ttg ggc aag caa ggg tac cag ctt aga cag gga gac aac 1008Gln Asp
Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335
ttt gaa gtg tgg gaa cga cct ctc tca ggc tta gcc tgg gct gta gct
1056Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala
340 345 350 atg ata aac cgg cag gag att ggt gga cct cgc tct tat acc
atc gca 1104Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr
Ile Ala 355 360 365 gtt gct tcc ctg ggt aaa gga gtg gcc tgt aat cct
gcc tgc ttc atc 1152Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro
Ala Cys Phe Ile 370 375 380 aca cag ctc ctc cct gtg aaa agg aag cta
ggg ttc tat gaa tgg act 1200Thr Gln Leu Leu Pro Val Lys Arg Lys Leu
Gly Phe Tyr Glu Trp Thr 385 390 395 400 tca agg tta aga agt cac ata
aat ccc aca ggc act gtt ttg ctt cag 1248Ser Arg Leu Arg Ser His Ile
Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 cta gaa aat aca atg
cag atg tca tta aaa gac tta ctt taa 1290Leu Glu Asn Thr Met Gln Met
Ser Leu Lys Asp Leu Leu 420 425 14429PRTHomo sapiens 14Met Gln Leu
Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg
Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25
30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu
35 40 45 Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser
Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met
Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys
Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly
Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg
Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly
Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro
Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155
160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser
165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala
Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp
Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu
Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile
Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp
Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro
Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe
Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280
285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser
290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala
Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu
Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser
Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile
Gly Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly
Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu
Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400
Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405
410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420
425
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